biostudies-literatureMa CNational Institute of Allergy and Infectious DiseasesArizona Biomedical Research CommissionNIAID NIH HHS1022-1030https://www.ebi.ac.uk/biostudies/studies/S-EPMC9017246biostudies-literatureUnknown8(5)The global COVID-19 pandemic underscores the dire need for effective antivirals. Encouraging progress has been made in developing small-molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (M^pro). However, the development of papain-like protease (PL^pro) inhibitors faces several obstacles. Nevertheless, PL^pro represents a high-profile drug target given its multifaceted roles in viral replication. PL^pro is involved in not only the cleavage of viral polyprotein but also the modulation of host immune response. In this study, we conducted a drug-repurposing screening of PL^pro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PL^pro inhibitors with IC₅₀ values ranging from 3.39 to 8.28 μM. The three hits showed dose-dependent binding to PL^pro in the thermal shift assay. In addition, tropifexor inhibited the cellular PL^pro activity in the FlipGFP assay with an IC₅₀ of 10.6 μM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells at noncytotoxic concentrations. Overall, tropifexor represents a novel PL^pro inhibitor that can be further developed as SARS-CoV-2 antivirals.ACS infectious diseasesDrug-Repurposing Screening Identified Tropifexor as a SARS-CoV-2 Papain-like Protease Inhibitor.PMC9017246ADHS18-198859AI157046R01 AI147325AI158775R01 AI158775AI147325R01 AI157046Ma CHu YChoza JWang YWang JfalseDrug-Repurposing Screening Identified Tropifexor as a SARS-CoV-2 Papain-like Protease Inhibitor.The global COVID-19 pandemic underscores the dire need for effective antivirals. Encouraging progress has been made in developing small-molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (M^pro). However, the development of papain-like protease (PL^pro) inhibitors faces several obstacles. Nevertheless, PL^pro represents a high-profile drug target given its multifaceted roles in viral replication. PL^pro is involved in not only the cleavage of viral polyprotein but also the modulation of host immune response. In this study, we conducted a drug-repurposing screening of PL^pro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PL^pro inhibitors with IC₅₀ values ranging from 3.39 to 8.28 μM. The three hits showed dose-dependent binding to PL^pro in the thermal shift assay. In addition, tropifexor inhibited the cellular PL^pro activity in the FlipGFP assay with an IC₅₀ of 10.6 μM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells at noncytotoxic concentrations. Overall, tropifexor represents a novel PL^pro inhibitor that can be further developed as SARS-CoV-2 antivirals.2022-01-01T00:00:00Z2022 May2024-12-03T17:35:54.068Z2024-12-03T17:35:54.068ZS-EPMC90172463540456410.1021/acsinfecdis.1c00629